In order for mammalian sperm to fertilize eggs, a number of incompletely defined changes must occur within the sperm that are collectively referred to as "capacitation". Recently, it was demonstrated that a central requirement for sperm capacitation is an efflux of cholesterol from the sperm membrane. In the mouse, cholesterol efflux stimulates an increase in sperm protein tyrosine phosphorylation, and blocking cholesterol efflux inhibits sperm capacitation. In this proposed research, we will attempt to answer the question, "How does the efflux of cholesterol from sperm membranes stimulate signaling events required for sperm capacitation?" We propose to test the hypothesis that the sperm protein caveolin-1, a cholesterol-binding and scaffolding protein, regulates signaling events during capacitation in response to reductions in sperm membrane cholesterol levels. The major goals of this research are to identify signaling proteins that interact with caveolin-1 in sperm, and to determine if this interaction with caveolin-1 changes during capacitation. Identification of caveolin- 1 interacting proteins will help define signaling pathways that are stimulated during capacitation, and will make future experiments to test the necessity for cholesterol efflux for sperm capacitation possible.